CN106279465A - The method extracting hyaluronic acid from Andrias davidianus Blanchard skin or mucus - Google Patents
The method extracting hyaluronic acid from Andrias davidianus Blanchard skin or mucus Download PDFInfo
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- CN106279465A CN106279465A CN201610667457.7A CN201610667457A CN106279465A CN 106279465 A CN106279465 A CN 106279465A CN 201610667457 A CN201610667457 A CN 201610667457A CN 106279465 A CN106279465 A CN 106279465A
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- hyaluronic acid
- andrias davidianus
- davidianus blanchard
- mucus
- enzymolysis
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
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- Biochemistry (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
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- Organic Chemistry (AREA)
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Cosmetics (AREA)
Abstract
The present invention discloses a kind of method extracting hyaluronic acid from Andrias davidianus Blanchard skin or mucus, it is characterised in that carry out the most in accordance with the following steps: by Andrias davidianus Blanchard skin or/and Andrias davidianus Blanchard mucus is homogenized at 0 ~ 10 DEG C；To homogenate in add 5 ~ 10 times of volume pH 7 ~ 8 0.01 ~ 0.1M phosphate buffer and with homogenate mass ratio be the compound enzyme of 0.1 ~ 1%, enzymolysis 12 ~ 48 h；Described compound enzyme is that the trypsin of the pronase of 3500 u/mg, the papain of 6000u/mg and 6000 u/mg is mixed than for 1:5:5 ratio in unit of activity；Enzymolysis solution pH after enzymolysis is adjusted to 4 ~ 5, stands 12h, 6000rpm and be centrifuged 20 min, collect supernatant；Supernatant is separated by the ultrafilter membrane that molecular cut off is 50000 Da, takes the liquid that ultrafilter membrane retains；1:1.5 ~ 3 ratio adds mass concentration 95% ethanol by volume, collects precipitation, lyophilization, obtains hyaluronic acid.
Description
Technical field
The present invention relates to the preparation method of a kind of hyaluronic acid, especially a kind of preparation technology is simple, yield and purity is high, matter
The measured method extracting hyaluronic acid from Andrias davidianus Blanchard skin or mucus.
Technical background
Hyaluronic acid i.e. hyaluronic acid (Hyaluronic acid, HA), is a kind of macromolecule straight-chain polysaccharide, by N-acetyl ammonia
Base glucose and D-Glucose aldehydic acid disaccharide unit repetitive structure composition, be distributed widely in the connective tissue of human body and animal,
The hyaluronic acid structure of separate sources is identical, only there are differences in relative molecular weight size.Research shows, hyaluronic acid has
High flexibility, plasticity, permeability and good biocompatibility, degradable biological material absorbable as one, exist
The field such as medicine, cosmetics has a wide range of applications.As hyaluronic acid theory water retention value may be up to 800ml/g, can be as natural
Moisture factor；Hyaluronic acid as medicinal application in ophthalmology Lens implantation, corneal transplantation, resisting glaucoma operation and treatment of arthritis
In.
At present, the main source of hyaluronic acid has two kinds: one is from animals such as cockscomb, people's umbilical cord, animal eyeball and Corii Sus domesticas
Tissue extracts；Another kind is to obtain from Streptococcus microorganism with the method for fermentation.As Lu Wen hero has delivered " pig in 1999
The preparation research of skin hyaluronic acid ", it is, with neutral protease, Corii Sus domestica is carried out enzymolysis to obtain hyaluronic acid, although yield etc. are relative
Other technology increases, but also can only achieve 0.14%.Owing to being limited by raw material and yield, annual output less than 2000kg,
The market demand can not be met far away.
Since the nineties in last century, Hunan, Hubei, Shaanxi and Guangxi all establish substantial amounts of Andrias davidianus Blanchard chartering cost enterprise,
Existing 500 ~ 6,000,000 tails of whole nation giant salamander culture scale at present.Andrias davidianus Blanchard body surface without squama, many glandular integumentarias, constantly divide in growth course
Secrete the mucus including various active material.But, up to now, also not about extracting hyaluronic acid from Andrias davidianus Blanchard skin or mucus
Relevant report.
Summary of the invention
The present invention is to solve the above-mentioned technical problem existing for prior art, it is provided that a kind of preparation technology is simple,
Rate and purity height, the measured method extracting hyaluronic acid from Andrias davidianus Blanchard skin or mucus of matter.
The technical solution of the present invention is: a kind of method extracting hyaluronic acid from Andrias davidianus Blanchard skin or mucus, and its feature exists
In carrying out the most in accordance with the following steps:
A. by Andrias davidianus Blanchard skin or/and Andrias davidianus Blanchard mucus is homogenized at 0 ~ 10 DEG C；
B. add in homogenate 5 ~ 10 times of volume pH 7 ~ 8 0.01 ~ 0.1M phosphate buffer and with homogenate mass ratio be 0.1 ~
The compound enzyme of 1%, enzymolysis 12 ~ 48 h；Described compound enzyme is the papain of the pronase of 3500 u/mg, 6000u/mg
Mix than for 1:5:5 ratio in unit of activity with the trypsin of 6000 u/mg；Enzymolysis solution pH after enzymolysis is adjusted to 4 ~ 5, quiet
Put 12h, 6000rpm and be centrifuged 20 min, collect supernatant；
C. supernatant is separated by the ultrafilter membrane that molecular cut off is 50000 Da, take the liquid that ultrafilter membrane retains；Press
Volume ratio 1:1.5 ~ 3 ratio adds mass concentration 95% ethanol, collects precipitation, lyophilization, obtains hyaluronic acid.
The present invention is to use three kinds of enzymes to be configured to compound protease in proportion Andrias davidianus Blanchard skin or the homogenate of Andrias davidianus Blanchard mucus are carried out enzyme
Solve, various enzyme interactings, hyaluronic acid can be made to separate with protein pilotaxitic texture (20 ~ 30 DEG C) enzymolysis at normal temperatures, make collection
Supernatant in remove in enzymolysis solution containing the higher hyaluronic acid of concentration, the ultrafiltration membrance filter through molecular cut off 50kDa
Enzyme and enzymatic hydrolysate, and with ethanol precipitate, i.e. can get purity >=96%(mass percent) hyaluronic acid, yield is up to 1%(matter
Amount percentage ratio), preparation technology is simple, and low cost, prepared hyaluronic acid relative molecular weight is 1.2 × 106Da, can be applicable to
The field such as cosmetics, medicine.
Detailed description of the invention
Embodiment 1:
A. Andrias davidianus Blanchard skin 5kg is homogenized at 0 ~ 10 DEG C；
B. in homogenate, add 0.05M phosphate buffer and the compound enzyme of 50g of 20 L pH 7 ~ 8, enzymolysis 30 h；Described multiple
Synthase is by pronase (3500 u/mg), papain (6000 u/mg) and trypsin 6000 u/mg) by living
Unit of force mixes than for 1:5:5 ratio；With food grade hydrochloric acid, the enzymolysis solution pH after enzymolysis is adjusted to 5, stand 12h, 6000rpm from
The heart 20 min, collects supernatant 13.5L；
C. supernatant is separated by the ultrafilter membrane that molecular cut off is 50000 Da, take liquid that ultrafilter membrane retains altogether
5L；Add mass concentration 95% ethanol precipitation, collect precipitation, lyophilization, obtain 51g desciccate.
Bitter-Muir carbazole method and Elson-Morgan method with Lu Wen hero report are respectively to the D-in desciccate
Glucuronic acid and 2-Acetamido-2-deoxy-D-glucose content detect, and find that D-Glucose aldehydic acid and 2-Acetamido-2-deoxy-D-glucose contain
Amount mol ratio is 1:1, and respective content reaches the 43% and 49% of gross mass, wherein contains only the protein of mass percent 0.07%,
And it is higher to use Intrinsic Viscosity Method to record prepared hyaluronic acid (desciccate) relative molecular weight, is 1.2 × 106Da。
Inspection in " preparation research of Corii Sus domestica hyaluronic acid " that the testing result of embodiment 1 and Lu Wen hero are delivered for 1999
Survey Comparative result and be shown in Table 1, it can be seen that use hyaluronic acid yield, purity and the quality that the method for the present invention produces all far above right
Ratio.
Table 1: embodiment 1 testing result contrasts with comparative example
| Protein content (%) | D-Glucose aldehydic acid (%) | Molecular weight | Yield (%) | |
| Comparative example | <0.11 | 40.2 | 9.5×105Da | 0.14 |
| Embodiment 1 | 0.07 | 43.0 | 1.2×106Da | 1.00 |
Embodiment 2:
A. Andrias davidianus Blanchard mucus 5kg is homogenized at 0 ~ 10 DEG C；
B. in homogenate, add 0.1M phosphate buffer and the compound enzyme of 50g, enzymolysis 40 h of 30L pH 7 ~ 8；Described compound
Enzyme is by pronase (3500 u/mg), papain (6000 u/mg) and trypsin 6000 u/mg) by vigor
Unit mixes than for 1:5:5 ratio；With food grade hydrochloric acid, the enzymolysis solution pH after enzymolysis is adjusted to 4, stands 12h, 6000rpm centrifugal
20 min, collect supernatant 14.5L；
C. supernatant is separated by the ultrafilter membrane that molecular cut off is 50000 Da, take liquid that ultrafilter membrane retains altogether
5.1L；Add mass concentration 95% ethanol precipitation, collect precipitation, lyophilization, obtain 52g desciccate.
Bitter-Muir carbazole method and Elson-Morgan method with Lu Wen hero report are respectively to the D-in desciccate
Glucuronic acid and 2-Acetamido-2-deoxy-D-glucose content detect, and find that D-Glucose aldehydic acid and 2-Acetamido-2-deoxy-D-glucose contain
Amount mol ratio is 1:1, and respective content reaches the 42.8% and 48.7% of gross mass, wherein contains only the egg of mass percent 0.06%
White matter, and it is higher to use Intrinsic Viscosity Method to record prepared hyaluronic acid (desciccate) relative molecular weight, is 1.2 × 106Da。
Inspection in " preparation research of Corii Sus domestica hyaluronic acid " that the testing result of embodiment 2 and Lu Wen hero are delivered for 1999
Survey Comparative result and be shown in Table 2, it can be seen that use hyaluronic acid yield, purity and the quality that the method for the present invention produces all far above right
Ratio.
Table 2: embodiment 2 testing result contrasts with comparative example
| Protein content (%) | D-Glucose aldehydic acid (%) | Molecular weight | Yield (%) | |
| Comparative example | <0.11 | 40.2 | 9.5×105Da | 0.14 |
| Embodiment 1 | 0.06 | 42.8 | 1.2×106Da | 1.00 |
Andrias davidianus Blanchard mucus 500g can be extracted every year according to an adult Andrias davidianus Blanchard to calculate, hyaluronic acid 5g can be produced, become with 500 ~ 6,000,000 tails
Year Andrias davidianus Blanchard calculating, can prepare hyaluronic acid 25000kg, greatly alleviate the demand and supply contraction.
Claims (1)
1. the method extracting hyaluronic acid from Andrias davidianus Blanchard skin or mucus, it is characterised in that carry out the most in accordance with the following steps:
A. by Andrias davidianus Blanchard skin or/and Andrias davidianus Blanchard mucus is homogenized at 0 ~ 10 DEG C；
B. add in homogenate 5 ~ 10 times of volume pH 7 ~ 8 0.01 ~ 0.1M phosphate buffer and with homogenate mass ratio be 0.1 ~
The compound enzyme of 1%, enzymolysis 12 ~ 48 h；Described compound enzyme is the papain of the pronase of 3500 u/mg, 6000u/mg
Mix than for 1:5:5 ratio in unit of activity with the trypsin of 6000 u/mg；Enzymolysis solution pH after enzymolysis is adjusted to 4 ~ 5, quiet
Put 12h, 6000rpm and be centrifuged 20 min, collect supernatant；
C. supernatant is separated by the ultrafilter membrane that molecular cut off is 50000 Da, take the liquid that ultrafilter membrane retains；Press
Volume ratio 1:1.5 ~ 3 ratio adds mass concentration 95% ethanol, collects precipitation, lyophilization, obtains hyaluronic acid.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610667457.7A CN106279465B (en) | 2016-08-15 | 2016-08-15 | The method of sodium hyaluronate is extracted from giant salamander skin or mucus |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610667457.7A CN106279465B (en) | 2016-08-15 | 2016-08-15 | The method of sodium hyaluronate is extracted from giant salamander skin or mucus |
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| CN106279465A true CN106279465A (en) | 2017-01-04 |
| CN106279465B CN106279465B (en) | 2019-02-12 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1063692A (en) * | 1991-01-31 | 1992-08-19 | 无锡市肉类厂 | From pork skin, extract hyaluronic manufacture method |
| CN105285202A (en) * | 2015-11-14 | 2016-02-03 | 普定县瑞鸿生产力促进有限责任公司 | Health tea and processing method thereof |
-
2016
- 2016-08-15 CN CN201610667457.7A patent/CN106279465B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1063692A (en) * | 1991-01-31 | 1992-08-19 | 无锡市肉类厂 | From pork skin, extract hyaluronic manufacture method |
| CN105285202A (en) * | 2015-11-14 | 2016-02-03 | 普定县瑞鸿生产力促进有限责任公司 | Health tea and processing method thereof |
Non-Patent Citations (3)
| Title |
|---|
| 吴梧桐等: "《生物化学（第3版）》", 31 August 2015, 中国医药科技出版社 * |
| 姚日生等: "《药用高分子材料（第2版）》", 30 April 2008, 化学工业出版社 * |
| 朱宝泉: "《生物制药技术（第1版）》", 30 June 2004, 化学工业出版社 * |
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